Helen M. Poppleton

Transmodulation of Epidermal Growth Factor Receptor Function by Cyclic AMP-dependent Protein Kinase

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Δ1022–1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRΔ1022–1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Δ1022–1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro andin vivo.

Stimulation of glycosaminoglycan synthesis in cultured human dermal fibroblasts by interleukin 1. Induction of hyaluronic acid synthesis by natural and recombinant interleukin 1s and synthetic interleukin 1 beta peptide 163-171.

Hyaluronic acid (HA) is believed to play a critical role in wound healing and in morphogenesis. Factors controlling the production of HA by fibroblasts in normal and pathological states are not completely understood. In this report we have observed that natural human interleukin (IL-1)1 beta and human recombinant (hrIL)-1 alpha and beta are potent stimulators of HA production by fibroblasts in vitro. Hyaluronic acid is the major species of glycosaminoglycan (GAG) stimulated by IL-1 in fibroblasts. PGE2 does not appear to be involved directly in this IL-1 effect on fibroblasts, but stimulation of HA production by IL-1 is dependent on protein synthesis. The synthetic human IL-1 beta peptide 163-171 (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), which has been previously shown to stimulate thymocyte proliferation but not fibroblast PGE2 production, is also able to stimulate fibroblast HA production. The synthesis and secretion of IL-1 by mononuclear phagocytes at sites of inflammation and immune reactions in vivo could potentially serve as a signal for fibroblasts to synthesize HA, which in turn could serve to facilitate and modulate reparative and immune processes by virtue of its ability to alter cell-cell, cell matrix, and cell-membrane receptor interactions.

The juxtamembrane, cytosolic region of the epidermal growth factor receptor is involved in association with alpha-subunit of Gs.

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsα is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsα we have investigated Gsα complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsα. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsα. However, interactions of these regions of the EGF receptor with constitutively active Gsα were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsα (1 mol of Gsα/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsα. However, neither the C-terminally truncated EGF receptor (Δ1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsα, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsα did not alter the association of Gsα with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsα poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsα by guanosine 5′-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsα and that upon activation of Gsα this association is decreased.

Subcellular localization and biological actions of activated RSK1 are determined by its interactions with subunits of cyclic AMP-dependent protein kinase.

ABSTRACT Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.

Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipatated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.

[21] Epidermal growth factor-mediated regulation of G proteins and adenylylcyclase in cardiac muscle

Publisher Summary This chapter presents a number of experimental approaches that have proved to be very useful in elucidating the mechanism(s) involved in epidermal growth factor (EGF) -mediated stimulation of cardiac adenylylcyclase. However, there are several additional questions pertaining to the detailed understanding of the mechanism(s) involved in interactions between the EGF receptor and G s interaction. Thus, the in vitro studies demonstrate the juxtamembrane region of the EGF receptor that is important for stimulation of G s by performing mutation(s) of key residue(s) in the juxtamembrane region of the EGF receptor, to determine whether the ability of EGF to stimulate adenylylcyclase can be obliterated. Similarly, it is important to determine the region(s) of G sα that interact with the EGF receptor. It is also determine that what elements confer specificity to the ability of EGF to stimulate adenylylcyclase. The latter goal poses a particularly important question, because EGF does not elevate cAMP content in all cells that express the EGF receptor, G s , and adenylylcyclase.